Diagnostic and therapeutic methods for EFMR (epilepsy and mental retardation limited to females)

ABSTRACT

Methods and kits for the diagnosis of illnesses related to protocadherin 19 (PCDH 19) protein deficiency or altered PCDH 19 protein function, in particular EFMR (Epilepsy and Mental Retardation limited to Females) are provided, as well as methods and kits for the identification of a predisposition to such illnesses and methods of screening subjects to identify carriers of such illnesses and methods and kits for the therapeutic or prophylactic treatment of PCDH 19 deficiency or altered PCDH 19 protein function. Further, nucleotide and amino acid sequences corresponding to a complete PCDH19 open reading frame (ORF), mutant sequences encoding non-functional PCDH19 mRNA or altered PCDH19 mRN A are described along with transformed cells and non-human transgenic animals comprising wild-type or mutant PCDH19 ORF nucleotide sequences.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a national stage of co-pending PCTApplication No. PCT/AU2009/000008, filed Jan. 5, 2009, which in turnclaims priority from U.S. Provisional Application Ser. No. 61/010,176,filed Jan. 4, 2008. Applicants claim the benefits of 35 U.S.C. § 120 and35 U.S.C. § 365 as to the PCT application, and 35 U.S.C. § 119(e) as tothe U.S. Provisional Application Ser. No. 61/010,176, and the entiredisclosures of both applications are incorporated herein by reference intheir entireties.

FIELD OF THE INVENTION

The present invention relates to nucleotide and amino acid sequencescorresponding to a complete protocadherin 19 (PCDH19) open reading frame(ORF), and mutant nucleotide sequences encoding non-functional oraltered PCDH19 mRNA or non-functional or altered PCDH19 protein whichcan result in illnesses related to PCDH19 protein deficiency or alteredfunction in human subjects, in particular EFMR (Epilepsy and MentalRetardation limited to Females).

BACKGROUND OF THE INVENTION

Inherited diseases caused by mutations on the X chromosome are generallycharacterised by the affected status of carrier males and sparing ofcarrier females. EFMR (Epilepsy and Mental Retardation limited toFemales) is a unique X-linked condition which, by contrast, sparescarrier males and is expressed in females (Ryan S G et al., 1997). EFMRis a rare condition characterised by seizure onset in early childhood(6-36 months) and cognitive impairment. The phenotype is restricted tofemales with males apparently spared, demonstrating normal cognitivefunction and absence of seizures.

Prior to the studies described herein, the cause of EFMR was unknown,with the presence of EFMR not previously attributed to any specificgenetic factor. The studies described herein now identify theprotocadherin 19 (PCDH19) gene as responsible for EFMR.

By the systematic re-sequencing of 737 X-linked genes, seven differentmutations in the PCDH19 gene were identified in seven unrelated familieswith EFMR. Five of these mutations result in the introduction of apremature termination codon resulting in non-functional PCDH19 mRNA thatis degraded by nonsense mediated decay (NMD) processes. The two othermutations have been determined to be missense mutations and are likelyto affect adhesiveness of the PCDH19 protein through impaired calciumbinding.

PCDH19 is the first cadherin to be implicated in epilepsy and mentalretardation. The expression analysis described herein shows a role forPCDH19 in normal neuronal development. A mechanism of phenotype rescuethat saves transmitting males (ie carrier males) from clinicallyexpressing the disorder is suspected, through a related male-specifichuman gene, protocadherin 11Y (PCDH11Y) (Blanco P et al., 2000). Thismechanism is consistent with the remarkable mode of inheritance observedin EFMR.

The studies described herein have identified nucleotide and amino acidsequences corresponding to a complete PCDH19 open reading frame (ORF) aswell as mutant sequences encoding non-functional PCDH19 mRNA ornon-functional PCDH19 protein. These are shown to be related toillnesses associated with PCDH19 protein deficiency or altered functionsuch as epilepsy and mental retardation, in particular EFMR. Further,male carriers of the PCDH19 deficient genotype have been shown to berescued from the disease phenotype by the male-specific protocadherinPCDH11Y.

The identification of the complete PCDH19 ORF and the identification ofmutations in the nucleotide sequence causing a disease state provide formethods for diagnosis of illnesses related to PCDH19 protein deficiencyor altered PCDH19 protein function, methods for the identification of apredisposition to such illnesses, methods of screening to identifycarriers of such illnesses methods, and agents for the therapeutic orprophylactic treatment of PCDH19 deficiency.

SUMMARY OF THE INVENTION

Thus, in a first aspect, the present invention provides a method ofdiagnosing an illness related to functional protocadherin 19 (PCDH19)protein deficiency or altered PCDH19 protein function, or assessing apredisposition to an illness related to functional PCDH19 proteindeficiency or altered PCDH19 protein function, or screening to identifycarriers of illnesses related to functional PCDH19 protein deficiency oraltered PCDH19 protein function, wherein said method comprises the stepof:

-   -   (i) detecting in a suitable biological sample from a subject, a        loss of PCDH19 protein function or altered PCDH19 protein        function.

In a second aspect, the invention provides a kit for diagnosing anillness related to functional protocadherin 19 (PCDH19) proteindeficiency or altered PCDH19 protein function, or assessing apredisposition to an illness related to functional PCDH19 proteindeficiency or altered PCDH19 protein function, or screening to identifycarriers of illnesses related to functional PCDH19 protein deficiency oraltered PCDH19 protein function, wherein said kit comprises one or moreof the following: an antibody or fragment thereof which specificallybinds to PCDH19 protein or polypeptide, or a fragment or variantthereof; and an oligonucleotide probe/primer molecule which specificallyhybridises to a polynucleotide molecule encoding PCDH19 protein orpolypeptide, or a fragment or variant thereof under high stringencyconditions.

In a third aspect, the present invention provides for the use of: apolynucleotide molecule comprising a nucleotide sequence showing atleast 70% sequence identity to a complete protocadherin 19 (PCDH19) openreading frame (ORF) nucleotide sequence according to SEQ ID NO: 1,wherein said nucleotide sequence encodes a functional PCDH19 protein orpolypeptide, or functional fragment or functional variant thereofencoded by a polynucleotide molecule comprising a nucleotide sequenceshowing at least 70% sequence identity to the complete PCDH19 ORFnucleotide sequence according to SEQ ID NO: 1; in the treatment ofPCDH19 protein deficiency or altered PCDH19 protein function in asubject.

In a fourth aspect, the present invention provides a method for thetherapeutic or prophylactic treatment of protocadherin 19 (PCDH19)protein deficiency or altered PCDH19 protein function in a subject,wherein said method comprises the step of:

-   -   (i) administering to said subject: a polynucleotide molecule        comprising a nucleotide sequence showing at least 70% sequence        identity to the complete protocadherin 19 (PCDH19) open reading        frame (ORF) nucleotide sequence according to SEQ ID NO: 1,        wherein said nucleotide sequence encodes a functional PCDH19        protein or polypeptide, or a functional fragment or functional        variant thereof; a functional PCDH19 protein or polypeptide, or        functional fragment or functional variant thereof encoded by a        polynucleotide molecule comprising a nucleotide sequence showing        at least 70% sequence identity to the complete PCDH19 ORF        nucleotide sequence according to SEQ ID NO: 1; and/or an agent        that compensates for the loss of PCDH19 protein function;        optionally in combination with a pharmaceutically-acceptable        carrier.

In a fifth aspect, the present invention provides an agent capable oftreating a deficiency in functional protocadherin 19 (PCDH19) protein oraltered PCDH19 protein function in a subject.

In a sixth aspect, the present invention provides a method foridentifying an agent capable of treating a deficiency in functionalprotocadherin 19 (PCDH19) protein or altered PCDH19 protein function,wherein said method comprises the steps of;

-   -   (i) providing a cell or animal comprising a polynucleotide        molecule comprising a mutant sequence of the PCDH19 ORF        nucleotide sequence shown as SEQ ID NO: 1;    -   (ii) contacting a test agent with said cell or administering a        test agent to said animal; and    -   (iii) comparing a response in said cell or animal with a control        response.

In a seventh aspect, the present invention provides a kit for use in themethod of the sixth aspect, wherein said kit comprises instructions forthe operation of the method together with one or more containers and/orvessels containing one or more cell(s) or animal(s) comprising apolynucleotide molecule comprising a mutant sequence of theprotocadherin 19 (PCDH19) ORF nucleotide sequence shown as SEQ ID NO: 1.

In an eighth aspect, the present invention provides a kit foridentifying an agent capable of treating a deficiency in functionalprotocadherin 19 (PCDH19) protein or altered PCDH19 protein function,wherein said kit comprises;

-   -   (i) a cell or animal comprising a polynucleotide molecule        comprising a mutant sequence of the PCDH19 ORF nucleotide        sequence shown as SEQ ID NO: 1; and optionally,    -   (ii) a control cell or animal comprising a polynucleotide        molecule comprising a wild-type form of the complete PCDH19 ORF        nucleotide sequence shown as SEQ ID NO: 1, said wild-type form        encoding a functional PCDH19 protein or polypeptide, or a        functional fragment or functional variant thereof.

In a ninth aspect, the present invention provides an isolated protein orpolypeptide comprising an amino acid sequence encoded by a nucleotidesequence showing at least 70% sequence identity to a completeprotocadherin 19 (PCDH19) ORF nucleotide sequence according to SEQ IDNO: 1, or a functional fragment or variant thereof.

In a tenth aspect, the present invention provides an isolatedpolynucleotide molecule comprising a nucleotide sequence showing atleast 70% sequence identity to a complete protocadherin 19 (PCDH19) ORFnucleotide sequence according to SEQ ID NO: 1 or a complementarysequence thereto.

In an eleventh aspect, the present invention provides a cell transformedwith the polynucleotide molecule of the tenth aspect.

In a twelfth aspect, the present invention provides a non-human animalcomprising the polynucleotide molecule of the tenth aspect.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows pedigrees of the seven EFMR families assessed in thestudies described herein. A specific mutation in the PCDH19 gene,responsible for EFMR in each family, is indicated alongside thecorresponding sequence chromatogram section showing the location of themutation. Females presenting with EFMR are represented by filled circlesand carrier males are represented by small circles within squares.

FIG. 2 shows a schematic diagram of the PCDH19 protein with the signalpeptide, extracellular cadherin (EC), transmembrane (TM) and cytoplasmic(CM) domains indicated. The relative locations of mutations found in theEFMR families are also shown.

FIG. 3 shows the partial alignment of the human PCDH19 with orthologuesof PCDH19 from other species and other human protocadherins. The highconservation of residues affected by two missense mutations, V441E (toppanel) and N557K (bottom panel) are indicated by rectangular boxes. Thecalcium ion-binding acidic residues are also indicated by a bracketagainst both alignments.

FIG. 4 shows Northern blot (Clontech Laboratories, Inc., Mountain View,Calif., United States of America) analyses of PCDH19 and PCDH11X/Y invarious human brains tissues. The position of the ˜9.8 kb PCDH19transcript is indicated by an asterisk, while the position of thesmaller ˜9.5 kb PCDH11X/Y mRNAs is shown by an arrowhead. The bracketsindicate either non-specific binding of the PCDH19 probe or PCDH19degradation products.

FIG. 5 shows a section of a nucleotide sequence chromatogram from anEFMR affected female, indicating the detection of a mutation, 253C>T, ingenomic DNA (gDNA) (top panel), the absence of the mutant sequence infibroblast cDNA (middle panel) and the presence of both the mutant andwild-type cDNA after the treatment of fibroblasts with cyclohexamide(bottom panel). The position of the mutation is boxed.

FIG. 6 shows the expression of PCDH19 in murine central nervous system(CNS) at 15.5 days postcoital (a-f) and postnatal day 2 (g-l). (a, b)are adjacent sections stained with Haematoxylin and Eosin and processedfor PCDH19 in situ, respectively. (c, d, e) are higher magnificationimages of the boxed regions in b. Arrowheads in c indicate PCDH19expressing cells within the cortex; the asterisk in e highlights thedorsolateral wall of the lateral ventricle. (g, h) are adjacent sectionsstained with Haematoxylin and Eosin and processed for PCDH19 in situ,respectively. (i) is a posterior brain section (to h) highlightingPCDH19 expression. (j, k, l) are higher magnification images of theboxed regions in (g, h, respectively). Cx/P, cortical plate; Hn,Hippocampal neuroepithelium; lv, lateral ventricle; Th, thalamus; Hy,hypothalamus; icf, intercerebral fissure; Ob, olfactory bulbs; Ne, nasalepithelium. Magnification bars (a, b, f-i) represent 200 μM, bars in(c-e; and j-l) represent 50 μM.

FIG. 7 shows a diagrammatic representation of the expected mechanismunderlying the inheritance of EFMR whereby PCDH11Y functionally rescuesPCDH19 mutations in transmitting males.

FIG. 8 shows expression profiles of PCDH19 (top panel), PCDH11Y (middlepanel) and PCDH11X (bottom panel) in various regions of adult humanbrain tissues.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to methods and kits for thediagnosis of illnesses related to PCDH19 protein deficiency or alteredPCDH19 protein function, methods and kits for the identification of apredisposition to such illnesses, methods of screening subjects toidentify carriers of such illnesses, and methods and kits for thetherapeutic or prophylactic treatment of PCDH19 deficiency or alteredPCDH19 protein function. The present invention also relates tonucleotide and amino acid sequences corresponding to a complete PCDH19ORF, mutant sequences encoding non-functional PCDH19 mRNA (eg which maybe degraded by nonsense mediated mRNA degradation (NMD) processes) oraltered PCDH19 mRNA, or non-functional PCDH19 protein or altered PCDH19protein causative of illnesses related to PCDH19 protein deficiency oraltered function in human subjects, and transformed cells and transgenicnon-human animals comprising wild-type or mutant PCDH19 ORF nucleotidesequences.

Thus, in a first aspect, the present invention provides a method ofdiagnosing an illness related to functional protocadherin 19 (PCDH19)protein deficiency or altered PCDH19 protein function, or assessing apredisposition to an illness related to functional PCDH19 proteindeficiency or altered PCDH19 protein function, or screening to identifycarriers of illnesses related to functional PCDH19 protein deficiency oraltered PCDH19 protein function, wherein said method comprises the stepof:

-   -   (i) detecting in a suitable biological sample from a subject, a        loss of PCDH19 protein function or altered PCDH19 protein        function.

Illnesses resulting from PCDH19 deficiency or altered PCDH19 proteinfunction include epilepsy and/or mental retardation, in particular EFMR.Thus, preferably, the method of the first aspect provides a method ofdiagnosing EFMR, assessing a predisposition to EFMR or screeningcarriers of EFMR, in particular, male carriers of EFMR. In particular,preferred methods include prenatal diagnosis or screening of EFMR.

The detection of a loss of PCDH19 protein function or altered PCDH19protein function can be used, in the case of a subject for which EFMRhas not previously been diagnosed, either on its own or in combinationwith other tests, to diagnose EFMR in the subject. For a subject inwhich EFMR has not previously been diagnosed and who is not showing anysigns of ill health due to EFMR, the detection of a loss of PCDH19protein function or altered PCDH19 protein function, can be used in anassessment of a predisposition to EFMR or carrier status of EFMR.

The detection of a loss of PCDH19 protein function or altered PCDH19protein function can involve one or more of detecting a mutant sequencein a PCDH19 ORF of the subject which encodes non-functional or alteredPCDH19 mRNA or non-functional or altered PCDH19 protein (eg bygenotyping the subject) or causes reduced expression of PCDH19 protein(eg mutations of the PCDH19 gene expression control sequences).Conveniently, this may be achieved by amplifying the PCDH19 nucleotidesequences (or a target region thereof) within a suitable biologicalsample and, thereafter sequencing the amplification product. Preferably,the detection of a loss of PCDH19 protein function involves detecting amutant sequence in the extracellular (EC) domain-encoding region of aPCDH19 ORF of the subject such as, for example, a mutant sequencecausing an amino acid substitution within or adjacent to a calciumion-binding site (eg within 20 amino acids of a calcium ion-bindingsite) such that calcium ion binding is impaired, or a mutant sequencecomprising a premature termination codon (PTC).

The detection of a loss of PCDH19 protein function or altered PCDH19protein function may also be indirectly achieved by conducting, forexample, assays for functional PCDH19 protein or polypeptide. Assays fordetecting functional PCDH19 protein or polypeptide, preferably comprisethe use of an antibody or fragment thereof that is capable ofspecifically binding with PCDH19 protein or polypeptide, or a functionalfragment or functional variant thereof, to determine the relative amountof the protein or polypeptide that is present in a suitable biologicalsample taken from the subject. This can involve the use of any of themethods well known to persons skilled in the art (eg standardELISA-based methods or in situ immunofluorescence using tissue sectionsamples). As such, the relative amount of functional PCDH19 protein orpolypeptide can be determined by quantitatively detecting the protein orpolypeptide with a specific antibody or fragment thereof (ie a primaryantibody) which is either directly conjugated to a detectable label oris otherwise detected via a secondary antibody or fragment thereofdirectly conjugated to a detectable label. Suitable detectable labelsinclude chromophores, fluorophores (eg fluorescein or FITC), radiolabels(eg ¹²⁵I), and enzymes such as horseradish peroxidase. These labels canbe used in methods and systems as are well known to persons skilled inthe art, which provide for the automation or partial automation of thestep of detecting the functional PCDH19 protein or polypeptide (eg by amicroplate reader or use of a flow cytometer). Generally, the relativeamount of functional PCDH19 protein will be determined by comparisonagainst the amount, or range of amounts, present in “normal samples” (egsamples from equivalent biological samples taken from normalsubject(s)).

Functional PCDH19 protein or polypeptide may be characterised as beingencoded by a nucleotide sequence showing at least 70% sequence identity,preferably at least 85% sequence identity, and, more preferably, atleast 95% sequence identity to a complete PCDH19 open reading frame(ORF) nucleotide sequence according to:

(SEQ ID NO: 1)atggagtcgc tcctgctgcc ggtgctgctg ctgctggcca tactgtggac gcaggctgcc 60gccctcatta atctcaagta ctcggtagaa gaggagcagc gcgccgggac ggtgattgcc 120aacgtggcca aagacgcgcg agaggcgggc ttcgcgctgg acccccggca ggcttcagcc 180tttcgcgtgg tgtccaactc ggctccacac ctagtggaca tcaatcccag ctctggcctg 240ctggtcacca agcagaagat tgaccgtgat ctgctgtgcc gccagagccc caagtgcatc 300atctcgctcg aggtcatgtc cagctcaatg gaaatctgcg tgataaaggt ggagatcaag 360gacctgaacg acaatgcgcc cagtttcccg gcagcacaga tcgagctgga gatctcggag 420gcagccagcc ctggcacgcg catcccgctg gacagcgctt acgatccaga ctcaggaagc 480tttggcgtgc agacttacga gctcacgccc aacgagctgt tcggcctgga gatcaagacg 540cgcggcgacg gctcccgctt tgccgaactc gtggtggaaa agagcctgga ccgcgagacg 600cagtcgcact acagcttccg aatcactgcg ctagacggtg gcgacccgcc gcgcctgggc 660accgttggcc ttagtatcaa ggtgaccgac tccaatgaca acaacccggt gtttagcgag 720tccacctacg cggtgagcgt gccagaaaac tcgcctccca acacacccgt catccgcctc 780aacgccagcg atccagacga gggcaccaac ggccaggtgg tctactcctt ctatggctac 840gtcaacgacc gcacgcgcga gctctttcag atcgacccgc acagtggcct ggtcactgtc 900actggcgctt tagactacga agaggggcac gtgtacgaac tggacgtgca ggctaaggac 960ttggggccca attccatccc ggcacactgc aaggtcaccg tcagcgtgct ggacaccaat 1020gacaatccgc cggtcatcaa cctgctgtca gtcaacagtg agcttgtgga ggtcagcgag 1080agcgcccccc cgggctacgt gatcgccttg gtgcgggtgt ctgatcgcga ctcaggcctc 1140aatggacgtg tgcagtgccg tttgctgggc aatgtgccct ttcgactgca ggaatatgag 1200agcttctcca ctattctggt ggacggacgg ctggaccgcg agcagcacga ccaatacaac 1260ctcacaattc aggcacgcga cggcggcgtg cccatgctgc agagtgccaa gtcctttacc 1320gtgctcatca ctgacgaaaa tgacaaccac ccgcactttt ccaagcccta ctaccaggtc 1380attgtgcagg agaacaacac gcctggcgcc tatctgctct ctgtgtctgc tcgcgacccc 1440gacctgggtc tcaacggcag tgtctcctac cagatcgtgc cgtcgcaggt gcgggacatg 1500cctgtcttca cctatgtctc catcaatccc aactcaggcg acatctacgc gctgcgatcc 1560tttaaccacg agcagaccaa ggcgttcgaa ttcaaggtgc tggccaagga cggcggcctt 1620ccctcactgc aaagcaacgc tacggtgcgg gtcatcatcc tcgacgtcaa cgacaacacc 1680ccggtcatca cagccccacc tctgattaac ggcactgccg aggtctacat accccgcaac 1740tctggcatag gctacctggt gactgttgtc aaggcagaag actacgatga gggcgaaaat 1800ggccgagtca cctacgacat gaccgagggc gaccgcggct tctttgaaat agaccaggtc 1860aatggcgaag tcagaaccac ccgcaccttc ggggagagct ccaagtcctc ctatgagctt 1920atcgtggtgg ctcacgacca cggcaagaca tctctctctg cctctgctct cgtcctaatc 1980tacttgtccc ctgctctcga tgcccaagag tcaatgggct ctgtgaactt gtccttgatt 2040ttcattattg ccctgggctc cattgcgggc atcctctttg taactatgat cttcgtggca 2100atcaagtgca agcgagacaa caaagagatc cggacctaca actgcagtaa ttgtttaacc 2160atcacttgtc tcctcggctg ttttataaaa ggacaaaaca gcaagtgtct gcattgcatc 2220tcggtttctc ccattagcga ggagcaagac aaaaagacag aggagaaagt gagcctaagg 2280ggaaagagaa ttgctgagta ctcctatggg catcaaaaga aatcaagcaa gaagaaaaaa 2340atcagtaaga atgacatccg cctggtaccc cgggatgtgg aggagacaga caagatgaac 2400gttgtcagtt gctcttccct gacctcctcc ctcaactatt ttgactacca ccagcagacg 2460ctgcccctgg gctgccgccg ctctgagagc actttcctga atgtggagaa ccagaatacc 2520cgcaacacca gtgctaacca catctaccat cactctttca acagccaggg gccccagcag 2580cctgacctga ttatcaacgg tgtgcctctg cctgagactg aaaactattc ttttgactcc 2640aactacgtga atagccgagc ccatttaatc aagagcagct ccaccttcaa ggacttagag 2700ggcaacagcc tgaaggatag tggacatgag gagagtgacc aaactgacag tgagcatgat 2760gtccagcgga gcctgtattg tgatactgct gtcaacgatg tgctgaacac cagtgtgacc 2820tccatgggat ctcagatgcc tgatcatgat cagaatgaag gatttcattg ccgggaagaa 2880tgccggattc ttggccactc tgacaggtgc tggatgcccc ggaaccccat gcccatccgt 2940tccaagtccc ctgagcatgt gaggaacatc atcgcgctgt ctattgaagc tactgctgct 3000gatgtcgagg cttatgacga ctgcggcccc accaaacgga ctttcgcaac ctttgggaaa 3060gatgtcagcg accacccggc tgaggagagg cctaccctga aaggcaagag gactgtcgat 3120gtgaccatct gcagccccaa ggtcaacagc gttatccggg aggcaggcaa tggctgtgag 3180gcgattagcc ctgtcacctc ccccctccac ctcaagagct ctctgcccac caagccttcc 3240gtgtcttaca ccattgccct ggctccccca gcccgtgatc tggagcagta tgtcaacaat 3300gtcaacaatg gccctactcg tccctctgaa gctgagcccc gtggagctga tagcgagaaa 3360gtcatgcatg aggtcagccc cattctgaag gaaggtcgca acaaagagtc ccctggtgtg 3420aagcgtctga aggatatcgt tctctaa. 3447

Most preferably, functional PCDH19 protein or polypeptide ischaracterised by comprising an amino acid sequence according to:

(SEQ ID NO: 2)MESLLLPVLLLLAILWTQAAALINLKYSVEEEQRAGTVIANVAKDAREAGFALDPRQASA 60FRVVSNSAPHLVDINPSSGLLVTKQKIDRDLLCRQSPKCIISLEVMSSSMEICVIKVEIK 120DLNDNAPSFPAAQIELEISEAASPGTRIPLDSAYDPDSGSFGVQTYELTPNELFGLEIKT 180RGDGSRFAELVVEKSLDRETQSHYSFRITALDGGDPPRLGTVGLSIKVIDSNDNNPVESE 240STYAVSVPENSPPNTPVIRLNASDPDEGTNGQVVYSFYGYVNDRTRELFQIDPHSGLVTV 300TGALDYEEGHVYELDVQAKDLGPNSIPAHCKVTVSVLDINDNPPVINLLSVNSELVEVSE 360SAPPGYVIALVRVSDRDSGLNGRVQCRLLGNVPFRLQEYESFSTILVDGRLDREQHDQYN 420LTIQARDGGVPMLQSAKSFTVLITDENDNHPHFSKPYYQVIVQENNTPGAYLLSVSARDP 480DLGLNGSVSYQIVPSQVRDMPVFTYVSINPNSGDIYALRSFNHEQTKAFEFKVLAKDGGL 540PSLQSNATVRVIILDVNDNTPVITAPPLINGTAEVYIPRNSGIGYLVIVVKAEDYDEGEN 600GRVTYDMTEGDRGFFEIDQVNGEVRTTRTFGESSKSSYELIVVAHDHGKTSLSASALVLI 660YLSPALDAQESMGSVNLSLIFIIALGSIAGILFVTMIFVAIKCKRDNKEIRTYNCSNCLT 720ITCLLGCFIKGQNSKCLHCISVSPISEEQDKKTEEKVSLRGKRIAEYSYGHQKKSSKKKK 780ISKNDIRLVPRDVEETDKMNVVSCSSLTSSLNYFDYHQQTLPLGCRRSESTFLNVENQNT 840RNTSANHIYHHSFNSQGPQQPDLIINGVPLPETENYSFDSNYVNSRAHLIKSSSTFKDLE 900GNSLKDSGHEESDQTDSEHDVQRSLYCDTAVNDVLNTSVTSMGSQMPDHDQNEGFHCREE 960CRILGHSDRCWMPRNPMPIRSKSPEHVRNIIALSIEATAADVEAYDDCGPTKRTFATFGK 1020DVSDHPAEERPTLKGKRTVDVTICSPKVNSVIREAGNGCEAISPVTSPLHLKSSLPTKPS 1080VSYTIALAPPARDLEQYVNNVNNGPTRPSEAEPRGADSEKVMHEVSPILKEGRNKESPGV 1140KRLKDIVL. 1148

On the other hand, assays for detecting loss of PCDH19 protein functionmay comprise the use of an antibody or fragment thereof that is capableof distinguishing between, for example, a wild-type PCDH19 protein orpolypeptide and a non-functional variant thereof. This may or may notresult in the identification of the particular form of the PCDH19protein or polypeptide that is present in the biological sample takenfrom the subject.

Otherwise, assays for detecting loss of PCDH19 protein function maycomprise determining the relative amount of messenger RNA (mRNA)encoding functional PCDH19 protein or polypeptide in a suitablebiological sample taken from the subject. The relative amount of mRNAencoding the protein or polypeptide may be determined by any of themethods well known to persons skilled in the art including Northern blot(by comparison to reference samples) and PCR-based mRNA quantificationmethods (eg using RT-PCR with primers conjugated to a detectable label).Generally, the relative amount of mRNA encoding the protein orpolypeptide will be determined by comparison against the amount, orrange of amounts, present in “normal samples” (eg samples fromequivalent biological samples taken from normal subject(s)).

Most preferably, the detection of a loss of PCDH19 protein functioncomprises detecting a mutant sequence encoding a non-functional variantof the PCDH19 amino acid sequence shown as SEQ ID NO: 2. Said mutantsequence may comprise any one or more of the nucleotide changes,relative to the nucleotide sequence shown as SEQ ID NO: 1, as follows:1322 T>A, 253 C>T, 2012 C>G, 2030_2031insT, 1671 C>G, 357delC and1091_1092insC. Methods for the detection of such nucleotide changes maycomprise the step of detecting any hybridisation of a suitableoligonucleotide probe/primer molecule under high stringency conditionsto the mutant sequence present in a suitable biological sample. Highstringency conditions are well known to persons skilled in the art, andare typically characterised by high temperature (ie high annealingtemperature) and low ionic strength (ie low salt concentration,especially of MgCl₂, KCl and NaCl). Thus, high stringency conditions mayvary according to the circumstances of the hybridisation (ie for probehybridisation, PCR amplification, etc). For the purposes of the presentinvention, the term “high stringency conditions” is to be understood asreferring to such conditions applicable to probe hybridisation (egconditions which: (1) employ low ionic strength and high temperature forwashing, for example, 15 mM NaCl/1.5 mM sodium citrate/0.1% NaDodSO₄ at50° C.; (2) employ, during hybridisation, a denaturing agent such asformamide, for example, 50% (vol/vol) formamide with 0.1% bovine serumalbumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphatebuffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C.; or(3) employ 50% formamide, 5×SSC (750 mM NaCl, 75 mM sodium citrate), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS and 10%dextran sulfate at 42° C. in 0.2×SSC (30 mM NaCl, 3 mM sodium citrate)and 0.1% SDS). An oligonucleotide molecule useful in the detection of amutant sequence according to the present invention may be suitable foruse as, for example, a probe or primer sequence. Typically, theoligonucleotide molecule will consist of 10 to 50 nucleotides and, morepreferably, about 15 to 30 nucleotides. Preferably, the oligonucleotidemolecule is derived from the nucleotide sequence shown as SEQ ID NO: 1or a complementary sequence thereto, or the nucleotide sequence as shownas SEQ ID NO: 1 but incorporating one or more of the nucleotide changesmentioned above (ie 1322 T>A, 253 C>T, 2012 C>G, 2030_2031insT, 1671C>G, 357delC and 1091_1092insC) or a complementary sequence thereto.

For the step of detecting a loss of PCDH19 protein function or alteredPCDH19 protein function, a suitable biological sample taken from thesubject may be selected from, for example, tissue biopsies and fixedsections (eg formalin fixed or paraffin embedded) or fixed cell samplesprepared therefrom, including epithelial samples, smear samples, bloodsamples, fecal samples, urine samples or buccal samples. The sample maybe pre-treated by, for example, filtration, separation or extractionmethods to partly or completely purify or isolate cells, proteins,polypeptides, polynucleotide molecules, oligonucleotide molecules orfragments thereof or fractions containing these components.

In a second aspect, the invention provides a kit for diagnosing anillness related to functional PCDH19 protein deficiency or alteredPCDH19 protein function, or assessing a predisposition to an illnessrelated to functional PCDH19 protein deficiency or altered PCDH19protein function, or screening to identify carriers of illnesses relatedto functional PCDH19 protein deficiency or altered PCDH19 proteinfunction, wherein said kit comprises one or more of the following: anantibody or fragment thereof which specifically binds to PCDH19 proteinor polypeptide, or a fragment or variant thereof; and an oligonucleotideprobe/primer molecule which specifically hybridises to a polynucleotidemolecule encoding PCDH19 protein or polypeptide, or a fragment orvariant thereof under high stringency conditions.

Such kits may comprise, for example, instructions for the operation ofthe method and, optionally, for thereafter diagnosing an illness relatedto functional PCDH19 protein deficiency or altered PCDH19 proteinfunction, or assessing a predisposition to an illness related tofunctional PCDH19 protein deficiency or altered PCDH19 protein function,or identifying carriers of illnesses related to functional PCDH19protein deficiency or altered PCDH19 protein function, together with oneor more containers or vessels containing said antibody or fragmentthereof and/or said oligonucleotide probe/primer molecule.

Preferably, said antibody or fragment thereof will bind to a protein orpolypeptide comprising an amino acid sequence showing at least 70%sequence identity to the PCDH19 amino acid sequence according to SEQ IDNO: 2. Further, preferably, said oligonucleotide probe/primer moleculewill hybridise to a polynucleotide molecule comprising a nucleotidesequence showing at least 70% sequence identity to the complete PCDH19ORF nucleotide sequence according to SEQ ID NO: 1.

In a third aspect, the present invention provides for the use of apolynucleotide molecule comprising a nucleotide sequence showing atleast 70% sequence identity to the complete protocadherin 19 (PCDH19)open reading frame (ORF) nucleotide sequence according to SEQ ID NO: 1,wherein said nucleotide sequence encodes a functional PCDH19 protein orpolypeptide, or a functional fragment or functional variant thereof; ora functional PCDH19 protein or polypeptide, or functional fragment orfunctional variant thereof encoded by a polynucleotide moleculecomprising a nucleotide sequence showing at least 70% sequence identityto the complete PCDH19 ORF nucleotide sequence according to SEQ ID NO:1; in the treatment of PCDH19 protein deficiency or altered PCDH19protein function in a subject.

Preferably, the said nucleotide sequence shows at least 85% sequenceidentity, and, more preferably, at least 95% sequence identity to thecomplete PCDH19 ORF nucleotide sequence according to SEQ ID NO: 1.

Most preferably, the said functional PCDH19 protein or polypeptidecomprises an amino acid sequence according to SEQ ID NO: 2.

For the sake of clarity, percentage levels of nucleotide sequenceidentity and amino acid sequence identity referred to herein are to beunderstood as meaning the “match percentage” calculated by the EMBL-EBIMultiple Alignment Using Fast Fourier Transform (MAFFT) tool using theBlosum 62 matrix (http://www.ebi.ac.uk/mafft/) and standard defaultsettings.

The term “functional fragment” as used herein is to be understood asreferring to a fragment which exhibits biological activity that issubstantially equivalent to a protein or polypeptide comprising thecomplete PCDH19 amino acid sequence shown as SEQ ID NO: 2.

The term “variant” as used herein in relation to an amino acid sequence,is to be understood as referring to a protein or polypeptide, orfragment thereof, comprising an amino acid sequence showing a high levelof sequence identity to the corresponding complete (or part thereof asthe case may be) of the amino acid sequence shown as SEQ ID NO: 2, butwhich includes one or more variations in the sequence which do notresult in any significant alteration of the biological activity of itsderivative protein or polypeptide (ie a protein or polypeptidecomprising the complete PCDH19 amino acid sequence shown as SEQ ID NO:2) or which otherwise results in enhanced or reduced biological activity(eg variants may include one or more amino acid substitutions, additionsor deletions, or may include the addition or deletion of a sequence ofamino acids, which enhances or reduces biological activity). A variantwith enhanced or reduced biological activity can therefore be regardedas a “functional variant”, whereas a variant which has no or minimalbiological activity can be regarded as a “non-functional variant”.Variations that do not result in any significant alteration of thebiological activity may include conservative amino acid substitutions.Exemplary conservative amino acid substitutions are provided in Table 1below. Particular conservative amino acids envisaged are: G, A, V, I, L,M; D, E; N, Q; S, T; K, R, H; F, Y, W, H; and P, Nα-alkylamino acids.

TABLE 1 Exemplary conservative amino acid substitutions ConservativeSubstitutions Ala Val*, Leu, Ile Arg Lys*, Gln, Asn Asn Gln*, His, Lys,Arg, Asp Asp Glu*, Asn Cys Ser Gln Asn*, His, Lys, Glu Asp*,γ-carboxyglutamic acid (Gla) Gly Pro His Asn, Gln, Lys, Arg* Ile Leu*,Val, Met, Ala, Phe, norleucine (Nle) Leu Nle, Ile*, Val, Met, Ala, PheLys Arg*, Gln, Asn, ornithine (Orn) Met Leu*, Ile, Phe, Nle Phe Leu*,Val, Ile, Ala Pro Gly*, hydroxyproline (Hyp), Ser, Thr Ser Thr Thr SerTrp Tyr Tyr Trp, Phe*, Thr, Ser Val Ile, Leu*, Met, Phe, Ala, Nle*indicates preferred conservative substitutions

In a fourth aspect, the present invention provides a method for thetherapeutic or prophylactic treatment of protocadherin 19 (PCDH19)protein deficiency or altered PCDH19 protein function in a subject,wherein said method comprises the step of:

-   -   (i) administering to said subject: a polynucleotide molecule        comprising a nucleotide sequence showing at least 70% sequence        identity to the complete protocadherin 19 (PCDH19) open reading        frame (ORF) nucleotide sequence according to SEQ ID NO: 1,        wherein said nucleotide sequence encodes a functional PCDH19        protein or polypeptide, or a functional fragment or functional        variant thereof; a functional PCDH19 protein or polypeptide, or        functional fragment or functional variant thereof encoded by a        polynucleotide molecule comprising a nucleotide sequence showing        at least 70% sequence identity to the complete PCDH19 ORF        nucleotide sequence according to SEQ ID NO: 1; and/or an agent        that compensates for the loss of PCDH19 protein function;        optionally in combination with a pharmaceutically-acceptable        carrier.

Preferably, the method comprises administering a functional PCDH19protein or polypeptide comprising an amino acid sequence according toSEQ ID NO: 2, or a functional fragment or functional variant thereof.

Alternatively or additionally, the method comprises administering anagent that compensates for the loss of PCDH19 function in the subject.Preferably, such an agent is a protein or polypeptide that compensatesfor PCDH19 function, such as another protocadherin/cadherin protein, ora functional fragment or functional variant thereof. An example of apreferred compensatory agent is PCDH11Y.

It is envisaged that the method of the fourth aspect may include theadministration of whole cells or recombinant delivery vehicles (eg viralvectors). Suitable polynucleotide molecule-delivery vectors includethose suitable for the chromosomal integration of the polynucleotidemolecule of the present invention (eg retroviral vectors), or simply forthe non-integrated expression of the polynucleotide molecule (eg plasmidvectors). Alternatively, the administration of a polynucleotide moleculeencoding a PCDH19 protein or polypeptide, or functional fragment orfunctional variant thereof, may involve any of the methods and/or agentsfor the delivery of “naked DNA” to cells well known to persons skilledin the art (eg liposomes, lipoplexes, polyplexes, gold microparticles,and conjugation to mannose and the like).

In a fifth aspect, the present invention provides an agent capable oftreating a deficiency in functional protocadherin (PCDH19) protein oraltered PCDH19 protein function in a subject.

Preferred agents according to the fifth aspect include: a polynucleotidemolecule comprising a nucleotide sequence showing at least 70% sequenceidentity, preferably at least 85% sequence identity, and, morepreferably, at least 95% sequence identity to the complete PCDH19 ORFnucleotide sequence according to SEQ ID NO: 1; or a functional PCDH19protein or polypeptide, or functional fragment or functional variantthereof encoded by a polynucleotide molecule comprising a nucleotidesequence showing at least 70% sequence identity, preferably at least 85%sequence identity, and, more preferably, at least 95% sequence identityto the complete PCDH19 ORF nucleotide sequence according to SEQ IDNO: 1. Most preferably, the agent is a polynucleotide moleculecomprising a nucleotide sequence encoding a PCDH19 protein orpolypeptide comprising the amino acid sequence shown as SEQ ID NO: 2; ora functional PCDH19 protein or polypeptide comprising the amino acidsequence shown as SEQ ID NO: 2.

Further preferred agents include an isolated or recombinantly expressedPCDH11Y protein or polypeptide, or a functional fragment or functionalvariant thereof. Homologues, analogues, orthologues or mimetics ofPCDH19 or PCDH11Y may also be suitable and, indeed, these may possessfurther desirable characteristics for use as therapeutic agents, forexample in vivo stability, safety and toxicity, pharmaceuticalacceptability and the like. The selection of preferred homologues,analogues, orthologues or mimetics of PCDH19 or PCDH11Y according todesirable characteristics may be readily determined by methods wellknown to persons skilled in the art.

Particularly preferred agents according to the fifth aspect are agentsthat are capable of providing treatment to EFMR or prophylactictreatment to subjects predisposed to EFMR.

Agents of the fifth aspect may be administered with a pharmaceuticallyacceptable carrier, and/or formulated into any suitablepharmaceutical/veterinary composition or dosage form (eg compositionsfor oral, buccal, nasal, intramuscular and intravenous administration).Typically, such a composition or dosage form will be administered to thesubject in an amount which is effective to treat EFMR or provideprophylaxis to a subject predisposed to EFMR, and may therefore beprovided at between about 0.01 and about 100 μg/kg body weight per dayof the agent, and more preferably, at between 0.05 and 25 μg/kg bodyweight per day of the agent. A suitable composition may be intended forsingle daily administration, multiple daily administration, orcontrolled or sustained release, as needed to achieve the most effectiveresult.

In a sixth aspect, the present invention provides a method foridentifying an agent capable of treating a deficiency in functionalprotocadherin 19 (PCDH19) protein or altered PCDH19 protein function,wherein said method comprises the steps of;

-   -   (i) providing a cell or animal comprising a polynucleotide        molecule comprising a mutant sequence of the PCDH19 ORF        nucleotide sequence shown as SEQ ID NO: 1;    -   (ii) contacting a test agent with said cell or administering a        test agent to said animal; and    -   (iii) comparing a response in said cell or animal with a control        response.

The method of the sixth aspect may identify agents capable of providinga treatment of illness caused by a deficiency in functional PCDH19protein or altered PCDH19 protein function, or which may be capable ofproviding a prophylactic treatment of functional PCDH19 proteindeficiency or altered PCDH19 protein function.

The control response referred to in step (iii) of the method may includea baseline response detected in said cell or animal without exposure tothe test agent or, alternatively, the control response may be a responsefollowing exposure to the test agent in cells or animals comprising anormal or wild-type complete PCDH19 ORF nucleotide sequence.

The test agent may be selected from known and novel compounds, complexesand other substances which may, for example, be sourced from private orpublicly accessible agent libraries (eg the Queensland Compound Library(Griffith University, Nathan, QLD, Australia) and the MolecularLibraries Small Molecule Repository (NIH Molecular Libraries, Bethesda,Md., United States of America). The test agent may therefore comprise aprotein, polypeptide or peptide (eg a recombinantly expressed PCDH19 orPCDH11Y protein or polypeptide, or a functional fragment or functionalvariant thereof), or a mimetic thereof (including so-called peptoids andretro-inverso peptides), but more preferably comprises a small organicmolecule and especially one which complies or substantially complieswith Lipinski's Rule of Five for “druglikeness” (Lipinski, C A et al.,2001). The test agent may also be selected on the basis of structuralanalysis of known or novel compounds or may otherwise be designedfollowing the further structural analysis of PCDH19 or PCDH11Y bindingsites, particularly calcium ion binding sites.

The method of the sixth aspect may be adapted for high-throughputscreening of large numbers of test agents.

The step of comparing a response in said cell or animal with a controlresponse may be conducted using one or more standard binding assayformats (eg ELISA-based or competition-based assays). Preferably, thetest agent will be labelled with a readily detectable label (eg afluorochrome or radioisotope) to allow detection of binding to, forexample, a calcium channel receptor. A change in activity may beobserved in such assays by using standard methods includingspectrophotometric, fluorimetric, calorimetric or chemiluminescent meanspreferably providing for the automation or partial automation of thedetecting step (eg by a microplate reader or use of a flow cytometer).

Preferred steps for comparing a response in an animal with a controlanimal (ie comprising a normal or wild-type complete PCDH19 ORFnucleotide sequence) involve the identification of a disease state insaid animal, in particular, the analysis of neurological indicators ofillness.

In a seventh aspect, the present invention provides a kit for use in themethod of the sixth aspect, wherein said kit comprises instructions forthe operation of the method together with one or more containers and/orvessels containing one or more cell(s) or animal(s) comprising apolynucleotide molecule comprising a mutant sequence of theprotocadherin 19 (PCDH19) ORF nucleotide sequence shown as SEQ ID NO: 1.

In an eighth aspect, the present invention provides a kit foridentifying an agent capable of treating a deficiency in functionalprotocadherin 19 (PCDH19) protein or altered PCDH19 protein function,wherein said kit comprises;

-   -   (i) a cell or animal comprising a polynucleotide molecule        comprising a mutant sequence of the PCDH19 ORF nucleotide        sequence shown as SEQ ID NO: 1; and optionally,    -   (ii) a control cell or animal comprising a polynucleotide        molecule comprising a wild-type form of the complete PCDH19 ORF        nucleotide sequence shown as SEQ ID NO: 1, said wild-type form        encoding a functional PCDH19 protein or polypeptide, or a        functional fragment or functional variant thereof.

The kit of the seventh or eighth aspect may further comprise means forcomparing a response in said mutant cell or animal with a controlresponse (eg as caused by a test agent), means for detecting a response(eg adhesiveness of PCDH19 or impaired calcium ion binding in the mutantcell or animal) and, for example, a test agent, and other components asare well known to persons skilled in the art including, for example,wash buffers, stabilisation buffers or other reagents.

In a ninth aspect, the present invention provides an isolated protein orpolypeptide comprising an amino acid sequence encoded by a nucleotidesequence showing at least 70% sequence identity to a completeprotocadherin 19 (PCDH19) ORF nucleotide sequence according to SEQ IDNO: 1, or a functional fragment or variant thereof.

As used herein, the term “isolated”, when used in relation to a proteinor polypeptide, or a functional fragment or variant thereof, or whenused in relation to a polynucleotide molecule, is to be understood asreferring to the protein, polypeptide, functional fragment, variant orpolynucleotide molecule in a form that is essentially free of wholecells, components thereof and/or other exogenous cellular or biologicalmaterials such as exogenous proteins, polypeptides, peptides and nucleicacid molecules. As such, an isolated protein, polypeptide, functionalfragment, variant or polynucleotide molecule, in accordance with thepresent invention, may be present in a preparation comprising no morethan 10% (by weight) of exogenous cellular or biological materials, andmay be prepared by any of the methods well known to persons skilled inthe art.

Preferably, the protein or polypeptide comprises an amino acid sequenceshowing at least 75% sequence identity to the complete PCDH19 amino acidsequence shown as SEQ ID NO: 2 and at least 65% sequence identity to theamino acid sequence corresponding to the extracellular cadherin (EC)domain.

More preferably, the protein or polypeptide, or functional fragment orvariant thereof, comprises an amino acid sequence showing at least 85%sequence identity and, still more preferably, at least 95% sequenceidentity to the complete PCDH19 amino acid sequence shown as SEQ ID NO:2. Most preferably, the protein or polypeptide comprises an amino acidsequence according to SEQ ID NO: 2.

In a preferred embodiment of the ninth aspect, the present inventionprovides a variant, preferably a non-functional variant, of the aminoacid sequence shown as SEQ ID NO: 2 including one or more amino acidmutations. Particularly preferred mutations included within such avariant include one or more amino acid mutations selected from thefollowing changes to the amino acid sequence shown as SEQ ID NO: 2;V441E, Q85X, S671X, L667fsX717, N557K, I119fsX122 and P364fsX375.

The isolated protein or polypeptide, functional fragment or variantthereof, may be isolated from tissues derived from whole organisms (egbiopsied tissues), from cultured tissues (eg cultured fibroblasts), orfrom other recombinant expression systems. This may involve any of themethods for isolating proteins or polypeptides well known to personsskilled in the art, including ion exchange, chromatographyelectrophoresis, isoelectric focusing, adsorption chromatography, paperchromatography, reverse-phase chromatography, hydrophobic interactionchromatography, dialysis, ultrafiltration, gel electrophoresis, gelfiltration, and ultracentrifugation.

Suitable techniques for expressing a recombinant protein or polypeptide,functional fragment or variant thereof according to the presentinvention are well known to persons skilled in the art and include, forexample, techniques for expressing recombinant His-tagged PCDH19 from asuitable expression vector or cassette using a suitable host cell (egCHO cells and BL21 cells). Thereafter, the His-tagged expressionproducts can be readily isolated using affinity chromatography (eg usinga Ni-NTA column (Qiagen Inc, Valencia, Calif., United States ofAmerica)). Where a functional fragment is to be provided, isolatedrecombinant protein or polypeptide may be cleaved using a proteolyticenzyme (eg trypsin).

Proteins, polypeptides, variants or, in particular, functional fragmentsaccording to the present invention may optionally be incorporated intosynthetic proteins or polypeptides such as fusion proteins. Fusionproteins may include components that assist in the production ordownstream processing (eg a protein, polypeptide, functional fragment orvariant thereof, may be linked to a secretory signal peptide, affinitypurification tag or the like).

In a tenth aspect, the present invention provides an isolatedpolynucleotide molecule comprising a nucleotide sequence showing atleast 70% sequence identity to the complete protocadherin 19 (PCDH19)ORF nucleotide sequence according to SEQ ID NO: 1 or a complementarysequence thereto.

Preferably, the polynucleotide molecule comprises a nucleotide sequenceshowing at least 85% sequence identity and, more preferably, at least95% sequence identity to the complete PCDH19 ORF nucleotide sequenceshown as SEQ ID NO: 1. Most preferably, the polynucleotide moleculecomprises a nucleotide sequence as shown as SEQ ID NO: 1.

A polynucleotide molecule of the present invention may encode a proteinor polypeptide comprising the amino acid sequence according to SEQ IDNO: 2, or a functional fragment or variant thereof. Alternatively, apolynucleotide molecule of the present invention may otherwise encode anon-functional PCDH19 mRNA (eg an mRNA including a premature terminationcodon which is degraded by NMD processes) or altered PCDH19 mRNA. Otherexamples of polynucleotide molecules according to the present inventionare oligonucleotide probe/primer molecules which consist of 10 to 50contiguous nucleotides and, more preferably, about 15 to 30 contiguousnucleotides of the complete PCDH19 ORF nucleotide sequence shown as SEQID NO: 1.

Where the polynucleotide molecule comprises a nucleotide sequenceshowing at least 70% sequence identity, preferably at least 85% sequenceidentity and, more preferably, at least 95% sequence identity to thenucleotide sequence shown as SEQ ID NO: 1, it will be appreciated thatsuch a polynucleotide molecule may vary from that nucleotide sequenceonly in minor changes which, for example, do not result in a significantalteration in an encoded protein or polypeptide due to degeneracy in theDNA code or which may be required in order to enhance expression in aparticular system (ie to comply with preferred codon usage). Further, itwill be appreciated that such a polynucleotide molecule may otherwiseencode a variant of a protein or polypeptide comprising the amino acidsequence shown as SEQ ID NO: 2 which shows enhanced or reducedbiological activity (eg a variant including one or more amino acidmutations in the extracellular cadherin (EC) domain and showing reducedadhesiveness through impaired calcium ion binding). Indeed, in anembodiment of the tenth aspect, the present invention provides apolynucleotide molecule encoding a variant, preferably a non-functionalvariant, including one or more amino acid substitutions, additions ordeletions in the extracellular cadherin (EC) domain and showing reducedadhesiveness through impaired calcium binding. Particularly preferredmutations encoded by such polynucleotide molecules include one or moreof the following changes to the amino acid sequence shown as SEQ ID NO:2; V441E, Q85X, S671X, L667fsX717, N557K, I119fsX122 and P364fsX375.With reference to the complete PCDH19 ORF nucleotide sequence shown asSEQ ID NO: 1, the mutations responsible for such amino acid sequencechanges may be, respectively; 1322 T>A, 253 C>T, 2012 C>G,2030_2031insT, 1671 C>G, 357delC and 1091_1092insC.

The polynucleotide molecule of the present invention may be used toexpress an encoded protein or polypeptide, or functional fragment orvariant thereof, by recombinant methodologies involving cloning of thepolynucleotide molecule into a suitable expression cassette or vectorand thereafter introducing the expression cassette or vector into asuitable host cell. Suitable expression vectors may include functionalsequences such as a multiple cloning site, a detection tag (egglutathione-S-transferase (GST) or green fluorescent protein (GFP)), atag for downstream purification (eg a histidine tag (His)), linker andfusion sequences.

In an eleventh aspect, the present invention provides a cell transformedwith the polynucleotide molecule of the tenth aspect.

The polynucleotide molecule may comprise a mutant sequence of the PCDH19ORF nucleotide sequence shown as SEQ ID NO: 1, and thereby encodenon-functional or altered PCDH19 mRNA, non-functional PCDH19 protein ora PCDH19 protein with altered function, or which otherwise causesreduced expression of PCDH19 protein, or a complementary sequencethereto.

The polynucleotide molecule may also consist or encode an antisense RNA,ribozyme, DNAzyme or interfering RNA molecule (eg siRNA) targeted toPCDH19.

The transformed cell may be selected from bacterial cells, insect cellsand mammalian cells. The cell may be transformed using any of themethods well known to persons skilled in the art including directuptake, transduction, f-mating or electroporation. The transformedpolynucleotide molecule may be maintained in a non-integrated form (egin a non-integrated plasmid expression vector), or alternatively, may beintegrated into the genome of the transformed cell.

The transformed cell can be employed in a variety of applications thatwill be readily apparent to persons skilled in the art, in particular,for the generation of an isolated recombinant protein or polypeptide, orfunctional fragment or variant thereof according to the presentinvention.

Where the transformed cell is intended for the production and harvest ofa PCDH19 protein or polypeptide, functional fragment or variant thereof,the expression of the recombinant product can be determined by, forexample, Western immunoblot assays for the direct detection of theprotein or polypeptide, functional fragment or variant thereof, or fordetection of an expression tag (eg a His tag).

In a twelfth aspect, the present invention provides a non-human animalcomprising the polynucleotide molecule of the tenth aspect.

The polynucleotide molecule may comprise a mutant sequence of the PCDH19ORF nucleotide sequence shown as SEQ ID NO: 1, and thereby encodenon-functional or altered PCDH19 mRNA, non-functional PCDH19 protein ora PCDH19 protein with altered function, or which otherwise causesreduced expression of PCDH19 protein, or a complementary sequencethereto.

The polynucleotide molecule may also consist or encode an antisense RNA,ribozyme, DNAzyme or interfering RNA molecule (eg siRNA) targeted toPCDH19.

The polynucleotide molecule is preferably uniformly integratedthroughout the animal's tissues. Where a chimeric animal is provided,the polynucleotide molecule is preferably present in cells of theanimal's nervous tissues.

The polynucleotide molecule may be introduced into the animal by any ofthe methods of transformation or transgenesis well known to personsskilled in the art. Such transformation methods include DNA transfection(via electroporation, liposome or protoplast fusion, or microinjection),infection with viral delivery vectors (ie vectors that facilitategenomic integration such as adenoviral and retroviral vectors), or viarandom mutagenesis followed by the selection of desired mutations byscreening. However, the animal of the present invention will generallybe preferably generated by micro-injection methodologies. To ensure thegenetic uniformity of resulting transgenic animals, micro-injection ispreferably performed at the one-cell embryo stage by any of the methodswell known to persons skilled in the art. Preferred transgenic animalsinclude rodents, in particular mice and rats.

The animals of the eleventh aspect can be employed in a variety ofapplications that will be readily apparent to persons skilled in theart, in particular, for use as in vitro or in vivo disease models foruse in methods or kits for screening potential agents to compensate forPCDH19 protein deficiency or altered PCDH19 protein function.

In a further aspect, the present invention provides an antibody orfragment thereof which specifically binds to the protein or polypeptide,functional fragment or variant thereof, of the ninth aspect.

Suitable antibodies include monoclonal and polyclonal antibodies.Suitable antibody fragments include fragments produced by enzymaticcleavage of antibodies such as Fab and F(ab′)₂ fragments, andrecombinant antibody fragments such as single chain Fv (scFv) fragments.

The antibody or fragment thereof may be capable of distinguishingbetween, for example, a wild-type PCDH19 protein (ie comprising thecomplete PCDH19 amino acid sequence shown as SEQ ID NO: 2) and a variantthereof, particularly, a non-functional variant thereof. Thus, thepresent invention extends to an antibody or fragment thereof thatspecifically binds to a variant of the complete PCDH19 amino acidsequence shown as SEQ ID NO: 2.

The present invention is hereinafter further described by way of thefollowing, non-limiting example and accompanying figures.

EXAMPLE

Materials and Methods

Patient and Family Details

Individuals from seven families with family members suffering fromepilepsy and mental retardation limited to females (EFMR) were admitted.The clinical details of Families 1-4 are described in Scheffer I E etal. (2007). Family 5 was screened on the basis of one sister havinginfantile seizures and Asperger syndrome and her sister having epilepsyand mild intellectual disability. The clinical details of members ofFamily 6 and Family 7 are described elsewhere (Juberg R C and Hellman CD, 1971; Fabisiak K and Erickson R P, 1990; and Ryan S G et al., 1997).

Northern Blotting

Human brain (MTN) blot II and V (acquired from Clontech Laboratories)were hybridised according to the manufacturer's instructions. Detectionassays utilised a probe containing nucleotides 2884-3257 of human PCDH19ORF (NCBI accession number 921478). The primers used to generate theprobe were:

(SEQ ID NO: 3) forward primer-5′CCGGATTCTTGGCCACTCTGAC3′; and(SEQ ID NO: 4) reverse primer-5′CAATGGTGTAAGACACGGAAG3′.

The 374 bp probe was labelled with radioactive α32P-dCTP (Perkin Elmer,Waltham, Mass., United States of America) using the Mega prime DNAlabelling system (GE Healthcare, Piscataway, N.J., United States ofAmerica).

RT-PCR Analyses

Total RNA was extracted from fibroblast cells using the RNeasy mini kit(Qiagen, Doncaster, VIC, Australia), and treated with DNase I (Qiagen).2 μg of RNA was primed with 1 μg of random hexanucleotides and subjectedto reverse-transcription for 90 minutes at 42° C. using Superscript IIaccording to the manufacturer's instructions (Invitrogen Corporation,Carlsbad, Calif., United States of America). The efficiency of thereaction was tested by PCR using primers specific to the ubiquitouslyexpressed ESD gene (Saviozzi et. al., 2006). cDNAs were amplified withTaq DNA polymerase (Roche, Basel, Switzerland) and specificsingle-stranded DNA primers for 35 cycles (denaturation, 94° C. for 30seconds; annealing, at specific Tm for each pair of primers for 30seconds; extension, 72° C. for 30 seconds). PCR products were separatedby agarose gel electrophoresis and stained with 1% ethidium bromide forvisualisation under UV.

Tissue Culture—Primary Skin Fibroblast Lines

A 3 mm skin biopsy excised from the upper arm of each subject was cutfinely and transferred to a tissue culture flask. The biopsy wascultured in RPMI medium with 20% foetal calf serum (FCS) (furthersupplemented with 4 mM L-Glutamine, 0.017 mg/ml benzylpenicillin) andgrown at 37° C. with 5% CO₂. Once established, fibroblasts wherecultured in RPMI with 10% FCS (also including the supplements describedabove).

Cycloheximide Treatment of Skin Fibroblast Cell Lines

Primary fibroblast cells were seeded 1×10⁴/cm² in RPMI with 10% FCS andincubated with 50 μg/ml cycloheximide (Sigma-Aldrich Co, St Louis, Mo.,United States of America), or media alone, for 6 hrs. Fibroblasts wereharvested using a sterile cell scraper (Techno Plastic Products AG,Trasadingen, Switzerland), then washed once in phosphate buffered saline(PBS) prior to total RNA extraction and reverse transcription togenerate cDNA as described above.

Mouse In Situ Hybridisation Analysis

15.5 days postcoital (dpc) embryonic heads and dissected postnatal day 2(P2) brains from c57xCBAF1 mice were fixed in 4% paraformaldehyde at 4°C., cryoprotected in 30% sucrose and frozen in optimal cuttingtemperature (OCT). In situ hybridisation of 16 μM sections was performedas described previously (Wilson L D et al., 2005) using adigoxygenin-labelled PCDH19 antisense RNA probe, prepared as previouslydescribed (Gaitan Y and Bouchard M, 2006). A total of three neonates andtwo embryos were analysed and representative sections were documented.No signal was detected in negative controls, which utilised a sensecontrol probe. Images were taken on a Zeiss Axiophot microscope,compiled and minimally processed (adjusted for colour and light/dark)using Adobe Photoshop CS®.

Semi-Quantitative RT-PCR

Gene expression profiles were generated using Rapid-Scan Gene ExpressionHuman Brain cDNA panels (Origene Technologies, Inc, Rockville, Md.,United States of America) containing first strand cDNA prepared frompolyA+ RNA. The cDNA panels allow semi-quantitative analysis due to thecDNAs being serially diluted over a 4-log range. The profiles wereobtained from panels containing approximately 1 ng of first strand cDNA.The PCR primer pair;

X-RT4F2—5′ GTA ACA AGT GTA CCT GGT ATG GAC T 3′ (SEQ ID NO: 5) and

X-RT5R2—5′ TCA ACC TTT ACT TTC ATC ACG 3′ (SEQ ID NO: 6), were used toamplify the PCDH11X sequence to yield a 683 bp product, and the primerpair;

Y-RT4F—5′ TAC AAC AAA CTG TCA CAA GTG TTT 3′ (SEQ ID NO: 7), and

Y-RT5R2—5′ TCA ACC TTT ACT TTC ATC ACA 3′ (SEQ ID NO: 8), were used toamplify PCDH11Y to yield a 681 bp product. The primers;

WLF—5′ AAC CAG AAT ACC CGC AAC AC 3′ (SEQ ID NO: 9) and

WLR—5′ CTG CAG ATG GTC ACA TCG AC 3′ (SEQ ID NO: 10), were used toamplify PCDH19 to yield a 626 bp product.

The PCR conditions used to amplify the PCDH19 product comprised aninitial step at 94° C. for 3 minutes, followed by 35 cycles at 94° C.for 30 seconds, 60° for 30 sec and 72° C. for 2 mins. The PCDH11X andPCDH11Y products were amplified using Hotstar Taq (Qiagen) according tothe Hotstar recommended cycling conditions (94° C. for 15 minutesfollowed by 10 cycles at [94° for 30 sec, 60° for 30 sec, 72° for 1 min]and then 30 cycles at [94° for 30 sec, 55° for 30 sec, 72° for 1 min]followed by 72° C. for 10 min).

GenBank Accession Numbers

Where available, partial nucleotide and amino acid sequences wereaccessed from the GenBank library. The accession numbers correspondingto these sequences are:

-   -   incomplete human PCDH19 mRNA accession number NM_020766.1;    -   incomplete human PCDH19 protein accession number NP_065817.1;        and    -   complete human PCDH19 mRNA and protein accession number GenBank        EF676096.

The GenBank library can be accessed at the following URLs;

NCBI: http://www.ncbi.nlm.nih.gov/, or Ensembl: http://www.ensembl.org/.

Nucleotide and amino acid sequences were determined by standard di-deoxychain termination sequencing methods as described in Sambrook, J.,Fritsch, E. F., and Maniatis, T., Molecular Cloning: A LaboratoryManual. Cold Spring Harbor Laboratory Press, NY, Vol 1, 2, 3 (1989).

Results and Discussion

Genetic Linkage Analysis and Identification of EFMR Gene

As a follow up to previous studies in which a single large Americanfamily with EFMR was described (Juberg R C and Hellman C D, 1971;Fabisiak K and Erickson R P, 1990; and Ryan S G et al., 1997), four newEFMR families were identified based on the inheritance pattern of EFMR,the electroclinical features of family members and the localisation ofthe gene responsible for EFMR (Scheffer I E et al., 2004; Scheffer I Eet al., 2007).

Pedigrees of the seven EFMR families in total were generated, whichshowed the characteristic inheritance pattern of affected females andtransmitting males (FIG. 1). Further linkage analysis within familiesshowed that the disease condition in each family consistently localisedto Xq22.

The EFMR gene was identified from re-sequenced 737 VEGA annotatedX-chromosome genes in probands from three families. In all threefamilies, each of the X chromosomes encoded protocadherin 19 (PCDH19)mutations (X^(m)) which were found to co-segregate with the EFMRclinical phenotype. The PCDH19 gene was known to be located at Xq22(Ensembl) within the original linkage region (Ryan S G et al., 1997). Anexample of a sequence chromatogram of a PCDH19 mutation as detected inan affected female from each family is shown alongside the pedigrees inFIG. 1.

Sequence analysis of family members showed a single nonsense nucleotidechange 2012C>G (residue S671X) in the PCDH19 gene in Family 3, whileFamilies 1 and 2 initially did not show any changes (FIG. 1). There wereno other deleterious nucleotide changes identified in the three familiesfor which the other 736 genes were screened. Subsequent comparativesequence analysis of the annotated PCDH19 open reading frame (ORF)(accession number NM_020766.1) revealed that it was incomplete. Thisprompted the sequencing of the additional N-terminal 1.5 kb of PCDH19ORF.

N-terminal sequencing of family members identified a missense change,1322T>A (residue V441E) in Family 1, a nonsense change, 253C>T (residueQ85X) in Family 2 and a putative frameshift change, 2030_2031 insT(residues L667fsX717) in Family 4. The N-terminal PCDH19 region inaffected females from an unreported Irish EFMR family (Family 5) wasalso sequenced resulting in the identification of a frameshift change,375delC (residues I119fsX122). The PCDH19 N-terminal region from theoriginal, large American EFMR family (Family 6) was re-sequencedresulting in the identification of a further frameshift mutation1091_1092insC (residue P364fsX375) (Juberg R C and Hellman C D, 1971;Fabisiak K and Erickson R P, 1990; Ryan S G et al., 1997) (FIG. 1).Finally, a nucleotide change 1671 C>G was identified in Family 7, codingfor an amino acid substitution of N557K.

Once aligned, silent nucleotide changes were further identified betweenfamily members and localised to positions 6 (G>A), 348 (G>A), 402 (C>A),1137 (C>T), 1627 (C>T) and 1683 (G>A) (frequencies shown in Table 2).

TABLE 2 Summary of the nucleotide changes found in PCDH19 (GenBankaccession number EF676096) with allele frequencies are indicated inparentheses. exon base change amino acid position 1 c.6G(99.8%) >A(0.2%) E2E 1 c.348G(99.8%) > A(0.2%) K116K 1 c.402C(97.1%) > A(2.9%)I134I 1 c.1137C(99.0%) > T(1.0%) G379G 1 c.1627C(68%) > T(32%) L543L 1c.1683G(99.8%) > A(0.2%) P561P

Partial alignment of the human PCDH19 amino acid sequence with otherhuman PCDH sequences and orthologues of PCDH19 from other speciesdemonstrates high conservation of residues affected by the two missensemutations, V441E and N557K, across other species and across otherfunctionally similar proteins (FIG. 3). Mutation V441E was observed inFamily 1 and N577K was observed in Family 7.

Accordingly, PCDH19 ORF nucleotide changes were identified in all sevenof the assessed EFMR families. All seven nucleotide changes segregatedwith the clinical phenotype in each EFMR family and were not identifiedin 250 male probands from families with putative X-linked mentalretardation (XLMR) or in 750 control X chromosomes. The positions of thePCDH19 mutations in conjunction with alignments showing the location andconservation of the two missense mutations further demonstrate thatPCDH19 mutation is causative of EFMR.

Relationship Between PCDH19 Mutation and Rett Syndrome or Autism

To determine whether PCDH19 mutations also contribute to thepresentation of disease conditions with similar phenotypes to EFMR,subjects presenting with Rett syndrome or autism, where no known causehad been determined, were tested for mutations at PCDH19.

Rett syndrome is a female specific disease known to present with asimilar phenotype to EFMR. 46 females with apparent Rett syndrome, whowere negative for mutations in the two Rett associated genes, MECP2 andCDKL5, were investigated. No nucleotide changes were found in the Rettsyndrome cohort, suggesting that PCDH19 mutations are unlikely tocommonly contribute to Rett syndrome.

Since autistic features were commonly seen in affected EFMR females, acohort of 55 females with autism and seizures were screened for changesin PCDH19. The absence of mutations in this cohort suggests that PCDH19mutations also do not commonly contribute to autism.

Characterisation of the PCDH19 Gene

The complete 3.447 kb ORF of the PCDH19 gene which consists of 6 exonswas annotated (GenBank accession number EF676096). The full-lengthprocessed PCDH19 mRNA is 9.765 kb long, this was confirmed by Northernblot analysis which showed a transcript size of approximately 9.8 kbusing a PCDH19 specific probe on combined male and female mRNA fromvarious areas of the adult human brain (FIG. 4, PCDH19 mRNA is indicatedby an asterisk). PCDH19 exon 2 was found to be alternatively spliced(results not shown).

PCDH19 encodes an 1148 amino acid protein belonging to the protocadherin(PCDH) δ2 subclass within the cadherin superfamily. The PCDH19 proteincontains a signal peptide, six extracellular cadherin (EC) repeats, atransmembrane (TM) domain and a cytoplasmic region with conserved CM1and CM2 domains. At FIG. 2, a schematic diagram is shown, illustratingthe locations of the PCDH19 amino acid sequence changes of each familywith respect to the signal peptide, the extracellular cadherin domain(comprising EC1, EC2, EC3, EC4, EC5 and EC6), the transmembrane domain(TM) and the cytoplasmic (CM1 and CM2) domains of the PCDH19 protein.All seven EFMR mutations identified are located in the largeextracellular domain.

The biological role of PCDH19 is not known; however, members of the PCDHfamily are predominantly expressed in the nervous system and arepostulated to be involved in the establishment of neuronal connectionsand in signal transduction at the synaptic membrane (Wu Q and ManiatisT, 1999; Yagi T and Takeichi M, 2000).

The partial alignments of human and orthologues of PCDH19 from otherspecies, shown at FIG. 3, shows high conservation of residues affectedby the two missense mutations, V441E (top panel) and N557K (bottompanel). The N557K mutation affects an invariant asparagine (N) residuewithin the EC5 domain (FIG. 2). The equivalent asparagine residue of EC1of classical cadherins (eg N100 of N-cadherin; Patel S D et al., 2006)and protocadherins (eg N101 of CNR/Pcdhα; Morishita H et al., 2006) isessential for calcium ion binding and for adhesive function of the EC1domain, thus it is expected that tissue mosaicism of PCDH19 negative andPCDH19 wild-type cells scrambles cell-cell communication manifestingclinically as EFMR. The valine residue at position 441 (EC4 in FIG. 2,or the equivalent of V96 of EC1 of N-cadherin; Patel S D et al., 2006;or V97 of EC1 of CNR/Pcdhα; Morishita H et al., 2006) is also highlyconserved (FIG. 3) and in close proximity to the calcium binding site(indicated by a bracket against both alignments). Thus, the two missensemutations, V441E and N557K are predicted to lead to loss of PCDH19function.

Thus, it is predicted that both PCDH19 missense variants adverselyaffect PCDH19 adhesive function through impaired calcium binding. Giventhe similarity of the clinical phenotype associated with all sevenPCDH19 mutations, it is reasonable to suggest that they all representloss of function mutations.

Stability of Mutant PCDH19 mRNA Transcripts

The PCDH19 mutations 253C>T, Q85X (Family 2) and 2012C>G, S671X (Family3) introduce a premature termination codon (PTC) into the PCDH19 mRNA.Such PTC-containing mRNAs are usually recognised by the NMD surveillancecomplexes and efficiently degraded (Maquat L E, 2004). The consequencesof the PCDH19 mutations 253C>T, Q85X (Family 2) and 2012C>G, S671X(Family 3) were examined on the stability of their respective mRNAs bydetecting PCDH19 mRNA in primary skin fibroblasts collected frombiopsied patients by RT-PCR.

FIG. 5 shows a sequence chromatogram from an EFMR affected female fromFamily 2 showing the detection of the mutation 253C>T, Q85X, in thegenomic DNA (gDNA) (top panel), the absence of the mutant sequence infibroblast cDNA (middle panel) and the presence of both mutant andwild-type cDNA after the treatment of fibroblasts with cyclohexamide(bottom panel), which inhibits the pioneer round of translation andleading to NMD. Similar results were found in tissues collected fromEFMR affected members of Family 3 (2012C>G mutation, S671X) (data notshown). The inhibition of NMD by cycloheximide treatment of skinfibroblast cells was found to preserve PTC mutation-containing mRNA.

To confirm that the absence of mutant PCDH19 mRNAs was not a consequenceof skewing of X-inactivation, random X-inactivation in DNA isolated fromblood and skin fibroblast cultures of each affected female availablewere assessed (data not shown). The absence of X inactivation skewing isin agreement with the published data (Ryan S G et al., 1997; Scheffer IE et al., 2007).

The results demonstrate that the PTC mutations in Families 2 and 3 leadto mRNA removal by NMD. It is anticipated that the mutations at2030_2031 insT (residues L667fsX717) found in Family 4, 375delC(residues I119fsX122) found in Family 5, and 1091_1092insC (residueP364fsX375) found in Family 6 will also lead to a complete loss offunctional mRNA as a consequence of NMD degradation of their respectivePTC-containing mRNA.

PCDH19 Expression in the Developing Brain

To investigate the expression of PCDH19 in the developing murine CNS, insitu hybridisation analysis PCDH19 mRNA in embryonic (15.5 days postcoitum (dpc)) and postnatal day 2 tissue was undertaken. FIG. 6 showsthe expression of PCDH19 in the developing murine CNS at 15.5 dpc (FIGS.6a to 6f ) and P2 (FIGS. 6g to 6l ). FIGS. 6a and 6b show adjacentsections stained with Haemotoxylin and Eosin and processed for PCDH19mRNA in situ, respectively. PCDH19 mRNA was expressed in a widespread,symmetrical pattern in the embryonic forebrain and frequently localisedto discrete cell clusters within the cortex (CxP, cortical plate),thalamus (Th) and hypothalamus (Hy). The lateral ventricle (lv) andhippocampal neuroepithelium (Hn) are also indicated. FIGS. 6c, 6d and 6eshow higher magnification images of the boxed regions in FIG. 6b . Thearrowheads in FIG. 6c indicate PCDH19-expressing cells within thecortex. In the cortex, expression was restricted to the cortical plateand extended medially into the intercerebral fissure (icf) (FIG. 6d ).The asterisk in FIG. 6e highlights the dorsolateral wall of the lateralventricle, robust expression was also detected in the ganglioniceminence that abuts the dorsolateral wall of the lateral ventricles.

At this stage, hippocampal expression was not observed on the medialedge of the lateral ventricle in the presumptive hippocampus (FIGS. 6band 6e ). However, subsequent analysis of anterior forebrain sectionsrevealed PCDH19 expression in the epithelial lining of the nasal cavity(consistent with a previous report (Gaitan Y and Bouchard M, 2006)) andin the olfactory bulbs (see FIG. 6f , olfactory bulbs indicated at Oband nasal epithelium at Ne).

FIGS. 6g and 6h show adjacent sections stained with Haemotoxylin andEosin and processed for PCDH19 mRNA by in situ hybridisation,respectively. FIG. 6i shows a brain section posterior with respect tothe brain section shown at FIG. 6h , each highlighting PCDH19 mRNAexpression. FIGS. 6j, 6k and 6l show higher magnification images of theboxed regions in FIGS. 6g and 6h , respectively.

At postnatal day 2, PCDH19 expression was maintained in discrete regionsof the cortex and the thalamus however, unlike the embryonic brain,expression was also observed in the hippocampus (FIGS. 6g, 6h , and 6i). In the cortex, expression was restricted to a band of cells thatspanned layers II-IV (indicated by arrows in FIGS. 6j and 6k ) whilstthe most prominent PCDH19 signal was observed in the CA1 and CA3 regionsof the hippocampus (FIGS. 6h and 6l ).

Consistent with previous Northern blot analyses (FIG. 2), PCDH19transcripts were not detected in white matter tracts including thecorpus callosum (FIG. 6h ). Together these data indicate that PCDH19 haswidespread expression in both the embryonic and adult brain includingthe developing cortex and hippocampus and are consistent with thefinding that mutation of this gene in humans is associated withcognitive impairment.

Mechanism for the Observed Disease Inheritance Patterns

Analysis of the EFMR family pedigrees showed that within the seven EFMRfamilies, there are 2 obligate carrier females (Family 6, individualIII.2 and Family 7, individual IV.15) who have not been diagnosed withEFMR, indicating the incomplete penetrance of the disorder. Havingidentified PCDH19 mutations, a mechanistic explanation was sought forthe remarkable inheritance pattern observed with EFMR.

One of the hypotheses, considered by Ryan S G et al., 1997, suggeststhat a dominant negative effect of mutant protein on wild-type proteinin females may be responsible for expression of the phenotype beinglimited to females. In this example, it has been demonstrated thatmutant PCDH19 mRNA is removed by NMD in affected females and a carriermale (FIG. 2 and data not shown), which is inconsistent with a dominantnegative hypothesis. However, in consideration of an alternativehypothesis (Ryan S G et al., 1997) involving a Y chromosome compensatorygene rescuing transmitting males from the EFMR phenotype, it was notedthat while there is no PCDH19 paralogue on the human Y chromosome, thereis the related protocadherin gene PCDH11Y within a block of X-Y homologyat Xq22. The PCDH11Y gene is thought to have arisen by transpositionfrom Xq after the divergence of chimpanzees and humans (Lambson B etal., 1992; Page D C et al., 1984) and therefore, the PCDH11Y gene isonly found in humans and in males.

A Northern blot analysis of PCDH19 and PCDH11X/Y was conducted in humancerebellum, cerebral cortex, medulla, spinal cord, occipital pole,frontal lobe, temporal lobe, putamen, amygdale, caudate nucleus, corpuscallosum, hippocampus, thalamus and whole brain tissues. FIG. 6c showsthe presence of PCDH19 transcripts (indicated by an asterisk,approximately 9.8 kb band), and the presence of PCDH11X/Y mRNAs(indicated by an arrowhead, approximately 9.5 kb band). FIG. 8 alsoshows expression profiles of PCDH19, PCDH11X and PCDH11Y in the adulthuman Frontal lobe, temporal lobe, cerebellum, hippocampus, substantialnigra, caudate nucleus, amygdale, thalamus, hypothalamus, pons, medulla,and spinal cord. Like many other members of the protocadherin family(Kim S Y et al., 2007) PCDH19, PCDH11Y and PCDH11X are expressed inhuman brain. PCDH11X and PCDH11Y show high sequence similarity, being98.1% identical at the nucleotide level and 98.3% identical at the aminoacid level (Blanco P et al., 2000) and show similar expression profilesin brain regions (Blanco P et al., 2000 and FIG. 8). However, PCDH11Xand PCDH11Y have undergone sequence divergence at the 5′ and 3′ ends oftheir ORF sequences, are regulated differently and show slightdifferences in their regions of expression in the brain (Blanco P etal., 2000). It is therefore possible that PCDH11X and PCDH11Y haveevolved different functions.

Sequence comparisons show that the extracellular cadherin (EC) domainsof both PCDH11X and PCDH11Y have some similarity to the EC domains ofPCDH19; a higher level of similarity than that seen between the ECdomains of PCDH19 and fellow PCDH d2 subclass members (PCDH-8, 10, 17and 18).

The high sequence identity and overlap in expression patterns betweenPCDH11X and PCDH11Y provides support for the hypothesis that PCDH11Xcompensates for PCDH19 loss of function mutations in females and thatboth PCDH11X and PCDH11Y compensate for PCDH19 mutations in males.

A uniquely evolved function of PCDH11Y may enable the protein to providegreater rescue of PCDH19 mutations than PCDH11X, which providesrationale for the greater frequency of spared male carriers than femalespresenting with EFMR.

A diagrammatic representation of the proposed mechanism underlying theinheritance of EFMR is illustrated in FIG. 7. The PCDH19 gene is locatedat Xq22.1 and is now known to harbour EFMR mutations. Within a homologyregion between the X and Y chromosomes there are two very similar PCDHgenes, PCDH11X on the X chromosome and PCDH11Y on the Y chromosome. Theresults provided herein indicate that PCDH11Y may functionally rescuePCDH19 mutations in transmitting males, while in females PCDH11X isunable to carry out rescue, explaining the EFMR phenotype being limitedto females.

The loss of function of all seven of the PCDH19 changes characterisedherein, their absence from control chromosomes, the absence of evidencefor potential disease-causing variants elsewhere on the X chromosome andthe mRNA studies conclusively show that the identified PCDH19 mutationsare causative of EFMR. The identification of nucleotide and amino acidsequences corresponding to a complete PCDH19 ORF provide for thedevelopment of diagnostic and therapeutic agents for EFMR and similardisorders associated with deficiencies in functional PCDH19 protein.Further, the elucidation of the suspected mechanism of PCDH19 rescue byPCDH11Y provides for the possibility of identifying and developingalternative therapeutic agents for the treatment of illnesses associatedwith PCDH19 protein-deficiency.

All seven of the characterised EFMR mutations are located in the largeextracellular domain and five of these are predicted to be complete lossof function mutations as a consequence of NMD degradation of theirrespective PTC containing mRNA. The remaining two missense mutations,V441E and N557K are predicted to lead to a loss of PCDH19 function. Lossof function may be the result of impaired calcium ion binding through alack of PCDH19 adhesiveness. Thus, genetic and functional targets areprovided for use in methods for diagnosis of illnesses associated withPCDH19 protein deficiency, methods for the identification of apredisposition to such illnesses and methods of screening to identifycarriers of such illnesses, and methods and kits for screening candidateagents for potential therapeutic use in the treatment of illnessesassociated with PCDH19 protein deficiency.

Although a preferred embodiment of the method of the present inventionhas been described in the foregoing detailed description, it will beunderstood that the invention is not limited to the embodimentdisclosed, but is capable of numerous rearrangements, modifications andsubstitutions without departing from the scope of the invention.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

All publications mentioned in this specification are herein incorporatedby reference. Any discussion of documents, acts, materials, devices,articles or the like which has been included in the presentspecification is solely for the purpose of providing a context for thepresent invention. It is not to be taken as an admission that any or allof these matters form part of the prior art base or were common generalknowledge in the field relevant to the present invention as it existedin Australia or elsewhere before the priority date of each claim of thisapplication.

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The invention claimed is:
 1. A method of detecting a mutation in aPCDH19 nucleic acid from a human subject, comprising: (a) contacting aPCDH19 nucleic acid from a biological sample obtained from a humansubject with a detectably labelled oligonucleotide that specificallyhybridizes to a PCDH19 nucleic acid sequence comprising a mutationselected from the group consisting of 1322T>A, 253C>T, 2012C>G,2030_2031insT, 1671C>G, 357delC and 1091_1092insC relative to SEQ ID NO:1, and (b) detecting hybridization of the oligonucleotide with thePCDH19 nucleic acid under high stringency conditions, wherein detectinghybridization is indicative of a mutation in the PCDH19 nucleic acid. 2.The method of claim 1, wherein the PCDH19 nucleic acid from thebiological sample comprises amplified DNA.
 3. The method of claim 1,wherein the oligonucleotide specifically hybridizes to 1322T>A relativeto SEQ ID NO:
 1. 4. The method of claim 1, wherein the oligonucleotidespecifically hybridizes to 253C>T relative to SEQ ID NO:
 1. 5. Themethod of claim 1, wherein the oligonucleotide specifically hybridizesto 2012C>G relative to SEQ ID NO:
 1. 6. The method of claim 1, whereinthe oligonucleotide specifically hybridizes to 2030_2031insT relative toSEQ ID NO:
 1. 7. The method of claim 1, wherein the oligonucleotidespecifically hybridizes to 1671 C>G relative to SEQ ID NO:
 1. 8. Themethod of claim 1, wherein the oligonucleotide specifically hybridizesto 357delC relative to SEQ ID NO:
 1. 9. The method of claim 1, whereinthe oligonucleotide specifically hybridizes to 1091_1092insC relative toSEQ ID NO: 1.